cells
Article
Generation and Characterization of Vascular Smooth
Muscle Cell Lines Derived from a Patient with
a Bicuspid Aortic Valve
Pamela Lazar-Karsten 1,† , Gazanfer Belge 2,† , Detlev Schult-Badusche 1 , Tim Focken 2 ,
Arlo Radtke 2 , Junfeng Yan 1 , Pramod Ranabhat 1 and Salah A. Mohamed 1, *
1
2
*
†
Department of Cardiac and Thoracic Vascular Surgery, University of Luebeck, D-23538 Luebeck, Germany;
[email protected] (P.L.-K.); [email protected] (D.S.-B.);
[email protected] (J.Y.); [email protected] (P.R.)
Center of Human Genetics and Genetic Counselling, University of Bremen, D-28359 Bremen, Germany;
[email protected] (G.B.); [email protected] (T.F.); [email protected] (A.R.)
Correspondence: [email protected]; Tel.: +49-451-500-6425; Fax: +49-451-500-2051
These authors contributed equally to this work.
Academic Editor: Alexander E. Kalyuzhny
Received: 6 September 2015; Accepted: 11 April 2016; Published: 21 April 2016
Abstract: Thoracic aortic dilation is the most common malformation of the proximal aorta and is
responsible for 1%–2% of all deaths in industrialized countries. In approximately 50% of patients
with a bicuspid aortic valve (BAV), dilation of any or all segments of the aorta occurs. BAV patients
with aortic dilation show an increased incidence of cultured vascular smooth muscle cell (VSMC)
loss. In this study, VSMC, isolated from the ascending aorta of BAV, was treated with Simian virus 40
to generate a BAV-originated VSMC cell line. To exclude any genomic DNA or cross-contamination,
highly polymorphic short tandem repeats of the cells were profiled. The cells were then characterized
using flow cytometry and karyotyping. The WG-59 cell line created is the first reported VSMC cell
line isolated from a BAV patient. Using an RT2 Profiler PCR Array, genes within the TGFβ/BMP
family that are dependent on losartan treatment were identified. Endoglin was found to be among the
regulated genes and was downregulated in WG-59 cells following treatment with different losartan
concentrations, when compared to untreated WG-59 cells.
Keywords: bicuspid aortic valve; vascular smooth muscle cell; WG-59 cell line; losartan
1. Introduction
Vascular smooth muscle cells (VSMCs) are of mesodermal origin and are present in various organs
(essential parts of the cardiovascular system, the respiratory system, the gastrointestinal tract and the
urinary tract). The VSMC phenotype is diverse [1]. In normal mature blood vessels, the predominant
phenotype is the quiescent or differentiated (contractile) phenotype, which regulates blood vessel
diameter and blood flow [2]. This phenotype can switch into the proliferative, migratory and synthetic
type, which is crucial for repair in response to injury and the synthesis of extracellular matrix proteins,
such as fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, transforming
growth factor beta (TGFβ), vascular endothelial growth factor and angiotensin II (AngII) [3]. VSMCs
are the main component of the aortic media, and their dysfunction plays an important role in many
different arterial diseases [4].
The loss of VSMCs is a characteristic of degenerative remodeling within the medial layer of
thoracic aortic aneurysms and dissection (TAAD) tissue. Moreover, patients with bicuspid aortic
valve (BAV) and aortic aneurysms exhibit VSMC alpha actin gene mutations (ACTA2, mapped to
chromosome 10q) [5,6]. In addition, a failure in switching from a synthetic to contractile VSMC
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phenotype generates intimal vascular lesions. VSMCs of patients with BAV are less mature than
those of tricuspid aortic valve (TAV) patients [7]. There is a significant difference between the aortic
wall structure in BAV patients compared to that in TAV patients, who present a thinner wall, lower
expression of typical maturation markers, such as SM22 α, calponin, α smooth muscle actin and nearly
no expression of smoothelin. BAV represents a high risk factor of TAAD. Patients with BAV are more
frequently affected by TAAD and at a younger age [8,9].
VSMCs of the aortic valve and ascending aorta share a common embryonic origin, the cardiac
neural crest [10,11]. Thus, analyzing the diseased ascending aorta can provide useful information
about malformations of the aortic valve. The dilation of the ascending aorta represents 43% of arterial
diseases with an upward trend. Due to the high mortality and lack of symptoms, it is a special
challenge to physicians, internists and cardiac surgeons. Since patients with BAV show increased
susceptibility to the development of ascending aortic dilation, studying the aortic wall structure may
render interesting insights. VSMC culture may provide useful help for this matter.
We used VSMCs isolated from a patient with BAV/TAAD and performed a Simian virus 40
(SV-40) transfection to generate an immortalized VSMC cell line. We then characterized these cells and
performed spectral karyotyping (SKY) to eliminate potential contamination by foreign DNA.
Since the TGFβ pathway plays an important role in the pathogenesis of TAAD, we analyzed
the effect of losartan on the expression of 84 genes related to the TGFβ pathway in VSMCs. The
disease Marfan syndrome (MFS), a connective tissue disorder, is caused by a mutation in the Fibrillin-1
gene. This mutation leads to overexpression of TGFβ signaling in MFS. Loeys–Dietz syndrome (LDS),
another disease with particularly strong predisposition for arterial aneurysm, shows decreased TGFβ
signaling. This leads to the assumption that TGFβ dysregulation may promote the aneurysmal process
in the aorta, thus arousing interest in further investigations of TGFβ. In the present study, the AngII
type 1 receptor (AT1 R) blocker losartan was used as a potential TGFβ signaling inhibitor. Losartan
reduces aortic growth and blunts TGFβ signaling in the aortic media of fibrillin-1-deficient mice,
thereby indicating an impact of the renin-angiotensin system in thoracic aortic aneurysm [12].
2. Material and Methods
2.1. Cell Culture of Primary VSMCs
A 46-year-old male patient was diagnosed with BAV and fusion of the right and non-coronary
cusps. He was admitted to the hospital to undergo aortic valve replacement due to Grade 3 pre-valve
stenosis. Ascending aorta tissue was removed during surgery; VSMCs were isolated out of the tunica
media for cell culture. The vascular smooth muscle sample was minced and treated with 0.26%
collagenase (250 U/mL, Serva; Heidelberg, Germany) at 37 ˝ C for 3–4 h. Following centrifugation, the
pellet was resuspended in culture medium (TC199 supplemented with Earle's balanced salt solution,
20% fetal bovine serum (FBS), 200 IU/mL penicillin and 200 µg/mL streptomycin and incubated at
37 ˝ C, 5% CO2 ). The monolayer culture was passaged by standard trypsin dispersion and resuspended
in culture medium.
2.2. Generating the WG-59 Cell Line from Primary VSMC Culture
To generate a human ascending aorta vascular smooth muscle cell line with an extended life span,
primary smooth muscle cells isolated from the biopsy material were transfected with a mammalian
expression vector containing genes encoding the SV-40 early region, according to Kazmierczak et al. [13].
Initial foci of transformed cells appeared 26 days post-transfection.
2.3. Fluorescence in Situ Hybridization Mapping of the SV-40 Early Region in VSMCs after Transfection
FISH studies were performed on pre-banded metaphase chromosomes from transformed muscle
cells. For hybridization, SV-40 plasmid DNA was used with DNA probes approximately 7.5 kb in
length, as described previously [13]. The hybridization procedure was performed according to the
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manufacturer’s instructions (Roche Diagnostics; Mannheim, Germany). The DNA probes were treated
with digoxigenin (DIG, Roche Diagnostics; Mannheim, Germany) and dissolved in hybridization
media followed by overnight incubation in a moist chamber at 37 ˝ C. Labeled probes were detected
using anti-digoxigenin–fluorescein isothiocyanate conjugates (FITC, Roche Diagnostics; Mannheim,
Germany). Chromosomes were counterstained with propidium iodide. In total, 20 metaphase events
were scored for analysis.
2.4. Immunofluorescence for Large T-Antigen in the WG-59 Cell Line
Large SV-40 T-antigen expression was analyzed according to Kazmierczak et al. [13].
Approximately 5 ˆ 104 VSMCs were plated onto cover slips and incubated in TC199 culture medium
supplemented with 20% (v/v) FBS for 48 h. Cells were washed with phosphate-buffered saline (PBS)
and fixed with methanol and acetone (10 min each at ´20 ˝ C). Cells were incubated for 30 min with
mouse anti-SV40 large T-antigen, washed in PBS and incubated with FITC-labeled goat anti-mouse
IgG (Merck Biosciences; Schwalbach, Germany) for another 30 min.
2.5. Chromosome Analysis with Spectral Karyotyping of the WG-59 Cell Line
For chromosome analysis, we used exponentially-growing cultures of transformed VSMCs. After
a 24-h growth period, metaphase chromosome spreads were prepared using colcemid (0.06 µg/mL for
40 min) in order to arrest the cultured cells during mitosis. A hypotonic solution (1:6 ratio of culture
medium in aqua bidestllata. and a fixative (3:1 ratio of methanol and acetic acid) were sequentially
applied. Finally, the chromosome suspension was dropped onto glass slides. The chromosomes were
GTG-banded according to routine techniques. The karyotype description followed the International
System for Human Cytogenetic Nomenclature 2013 (ISCN). SKY was performed according to the
manufacturer’s protocol (Applied Spectral Imaging (ASI), Migdal Ha’Emek, Israel). To describe the
karyotype, 12 metaphase spreads were acquired with an Axioskop 2 plus microscope (Carl Zeiss,
Göttingen, Germany) and analyzed using the SkyView Software. A composite karyotyping was done
according to ISCN 2013.
2.6. Highly Polymorphic Short Tandem Repeat Profiling
DNA was isolated from patient tissue and WG-59 using the QIAamp DNA mini kit (Hilden,
Germany) according to the manufacturer’s protocol. DNA concentration was measured using the
NanoDrop instrument (Wilmington, NC, USA). To screen the cells against cross-contamination and in
order to authenticate them as a human cell line originating from the donor’s material as the donor
tissue, short tandem repeat (STR) typing was performed. DNA profiling was carried out using
8 different and highly polymorphic STR loci. Furthermore, we tested the samples for the presence
of mitochondrial DNA sequences from rodent cells, such as mouse, rat, Chinese and Syrian hamster,
according to the manufacturer’s protocol (DSMZ, Braunschweig, Germany).
2.7. Characterization of the WG-59 Cell Line
The WG-59 cell characterization was performed by flow cytometry at the 20th passage. In brief,
100 µL of 1 ˆ 106 cells were incubated for 20 min at 4 ˝ C in BD Cytofix/Cytoperm (BD Bioscience,
Heidelberg, Germany) in order to fix and the cells and permeabilize the cell membrane. Cells were
then washed with 10% BD Wash/Perm Solution (BD Bioscience) and incubated with antibodies against
α-actin (R&D Systems, Minneapolis, MN, USA), myosin heavy chain (R&D Systems, Minneapolis,
MN, USA), calponin (Acris, San Diego, CA, USA) and smoothelin (Acris, San Diego, CA, USA) in a
total volume of 50 µL for 30 min. Cells were washed again in 10% BD Wash/Perm Solution and were
resuspended in 300–400 µL BD Staining Buffer (BD Bioscience). The measurement was performed on
a benchtop analyzer LSR II (Becton Dickenson, Heidelberg, Germany). FlowJo software (Tree Star,
Inc., Ashland, OR, USA) was used for data analysis. To exclude non-specific binding, mouse IgG1 and
IgG2a isotype controls (BioLegend, London, UK) were used.
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2.8. Collagen-Based Cell Contraction Assay
The contractility of the WG-59 cells at a passage of 50 was tested by subjecting the cells to a
collagen-based contraction analysis, using the cell contraction assay (Cell Biolabs Inc. San Diego, CA,
USA) according to the manufacturer’s protocol. Here, the cells were suspended in the DMEM media
provided, with a density of 5 ˆ 106 cells per mL. The collagen lattice was prepared by mixing 1 part of
the cell suspension with 4 parts of cold collagen gel solution (prepared with the supplied kit solutions).
To polymerize the collagen matrix, 0.5 mL of the cell-collagen mixture were then added to each well in
a 24-well plate and incubated for 1 h at 37 ˝ C. After polymerization, 1 mL of the culture medium was
added to each collagen gel lattice. The cell culture was incubated for 2 days. To initiate contraction, the
collagen gel was gently released from the side of the culture dishes with a sterile spatula. The size of
the matrix was measured at the beginning and end of the assay.
2.9. TGFβ Pathway and Data Analysis
The WG-59 cell line was grown to 80% confluency and incubated in serum-free media for 24 h.
Losartan (1 µM) was added to the cells for 24 h. Cellular RNA was extracted with TRIzol (Life
Technologies, Darmstadt, Germany). RNA was reverse transcribed into cDNA using theSuperScript
II-Kit (Life Technologies, Darmstadt; Germany), and expression of 84 genes related to the TGFβ
pathway was quantified using the RT2 Profiler PCR Array according to the manufacturer’s protocol
(Qiagen, Hilden, Germany). Three independently-treated samples and three controls were measured
using the RT2 Profiler PCR Array.
Data analysis was performed with RT2 Profiler PCR Array Data Analysis Version 3.5. The sample
results were normalized to GAPDH and α-actin. Samples treated with losartan were compared to
cells without treatment (control group). Genes with at least a two-fold change in expression and a
p-value < 0.05 were considered significantly regulated.
2.10. Single Gene Expression Analysis
To confirm the TGFβ pathway analysis results and to further investigate the effect of losartan
in various concentrations, the expression of endoglin and CDC25A was measured by single real-time
RT-PCR. SYBR Green was used to detect expression changes dependent on different losartan
concentrations (1, 10 and 100 µM) and compared to a non-treated control cell culture. As an internal
control for normalization, 18S RNA was used. Gene expression analysis was performed using
the Applied Biosystems 7000 Real-Time PCR System (Applied Biosystems by Life Technologies,
Darmstadt, Germany).
3. Results
3.1. Generating and Characterizing the WG-59 Cell Line
The WG-59 cell line was generated from VSMCs of the tunica media of an ascending aorta.
Immortalization was performed after SV-40 transfection. Initial foci of transformed cells appeared
26 days post-transfection (Figure 1A). Cells showed altered morphology, smaller cell size, loss of
contact inhibition, foci formation and T-antigen expression (Figure 1B). Transformed cells showed
higher proliferation capacity when compared to non-transformed cells. While non-transformed cells
had a maximum lifetime of 15 passages, we were able to cultivate the transformed cells up to the 70th
passage. Changes of the morphology and proliferation were monitored daily under the microscope
(Figure 1C).
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Figure
cells 26
26 days
days post-transfection
post-transfection in
in the
the bright
bright field
field microscope.
microscope.
Figure 1.
1. (A)
(A) Initial
Initial foci
foci of
of transformed
transformed cells
Foci
are
clearly
distinguishable
among
the
cultured
and
transfected
cells.
(B)
GFP-labeled
Foci are clearly distinguishable among the cultured and transfected cells. (B) GFP-labeled T-antigen
T-antigen
expression
transfected
cells. cells.
(C) Immortalized
confluent confluent
WG-59 in comparison
non-transfected,
expression inin
transfected
(C) Immortalized
WG-59 inwith
comparison
with
primary
confluentprimary
VSMCs;confluent
10-fold enlargement
(D). (E)
WG-59 cell(D).
culture,
third day
20. Passage;
non-transfected,
VSMCs; 10-fold
enlargement
(E) WG-59
cellofculture,
third
10-fold
(F) WG-59
cell culture,(F)
sixth
day of
20.culture,
Passage;
10-fold
day of enlargement.
20. Passage; 10-fold
enlargement.
WG-59
cell
sixth
dayenlargement.
of 20. Passage; 10-fold
enlargement.
3.2. Cytogenic Characteristics and DNA Profile of the WG-59 Cell Line
3.2. Cytogenic Characteristics and DNA Profile of the WG-59 Cell Line
To cytogenetically characterize the cell line, we investigated 60 metaphases (55th passage) where
we found
the largest (92) characterize
and the least the
(70) cell
number
chromosomes
1). The average
of
To cytogenetically
line,ofwe
investigated(Table
60 metaphases
(55thnumber
passage)
chromosomes
per
metaphase
is
76.6,
so
each
chromosome
should
be
present
in
three
to
four
copies.
where we found the largest (92) and the least (70) number of chromosomes (Table 1). The average
We
karyotyped
17 of the 60per
metaphases
and
losschromosome
of both Y-chromosomes
in ten metaphases.
number
of chromosomes
metaphase
is found
76.6, sothe
each
should be present
in three to
In
thecopies.
other five
one Y-chromosome
present.
In addition
to different
four
We karyotyped
karyotypedmetaphases,
17 of the 60only
metaphases
and found was
the loss
of both
Y-chromosomes
in
translocations
and
deletions
of
several
chromosomes,
the
loss
of
autosomal
chromosomes
2,
3,
4,
8,
ten metaphases. In the other five karyotyped metaphases, only one Y-chromosome was present. In
9,
10, 13, to
15,different
17, 18, 19,translocations
20 was observed
most metaphases.
The spectral karyotyping
a
addition
and in
deletions
of several chromosomes,
the loss of revealed
autosomal
pseudotriploid
a chromosome
of observed
70–91. Several
aberrations
were
present
in
chromosomes 2,karyotype
3, 4, 8, 9, with
10, 13,
15, 17, 18, 19,count
20 was
in most
metaphases.
The
spectral
duplicates
and
clonal numbers.
Single cellkaryotype
aberrations
could
be detected incount
nearlyof
every
metaphase
karyotyping
revealed
a pseudotriploid
with
a chromosome
70–91.
Several
(Figure
2). were present in duplicates and clonal numbers. Single cell aberrations could be detected
aberrations
in nearly every metaphase (Figure 2).
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Table 1. Karyotype of the examined immortalized cell culture showing several aberrations in different
chromosomes.
spectral
Table 1.SKY,
Karyotype
of karyotyping.
the examined immortalized cell culture showing several aberrations in
different chromosomes. SKY, spectral karyotyping.
Composite Karyotype SKY:
Composite Karyotype SKY:
70-91,XX,-Y[11],-Y[12],der(2)t(2;6)x2[11],-3[3],-4[4],der(6)t(6;15)[2],der(6)t(6;15der(8)t(9;8;2)x2[12],der(8)t(8;18)[7],
70-91,XX,-Y[11],-Y[12],der(2)t(2;6)x2[11],-3[3],-4[4],der(6)t(6;15)[2],der(6)t(6;15der(8)t(9;8;2)x2[12],de
der(8)t(8;18)x2[5],-9[4],+9[2],der(9)t(5;9)[5],der(9)t(5;9)x2[6],-10[4],-11[3],der(11)t(8;11)x2[7],der(12)t(5;12)x2[4],
r(8)t(8;18)[7],der(8)t(8;18)x2[5],-9[4],+9[2],der(9)t(5;9)[5],der(9)t(5;9)x2[6],-10[4],-11[3],der(11)t(8;11)x
-13[7],-13[3],rob(13;14)[4],rob(13;14)x2[7],-14[5],-14[7],der(14)t(14,19)[4],der(14)t(14,19)x2[4],-15[12],-15[12],
2[7],der(12)t(5;12)x2[4],-13[7],-13[3],rob(13;14)[4],rob(13;14)x2[7],-14[5],-14[7],der(14)t(14,19)[4],der(1
der(15)t(15;16)x2[9],-16[5],
4)t(14,19)x2[4],-15[12],-15[12], der(15)t(15;16)x2[9],-16[5],
del(16)x2[8],der(16)t(16;20)x2[3],-17[4],-18[11],-18[7],der(18)t(5;18)[2],der(18)t(11;18)x2[7],-19[2]-19[10],-20[3],
del(16)x2[8],der(16)t(16;20)x2[3],-17[4],-18[11],-18[7],der(18)t(5;18)[2],der(18)t(11;18)x2[7],-19[2]-19[1
-20[2],der(20)t(20;22)x2[9],-21[3],-22[12],-22[12][cp12]
0],-20[3],-20[2],der(20)t(20;22)x2[9],-21[3],-22[12],-22[12][cp12]
Figure 2. Illustration of detected chromosomes in WG-59 immortalized cultured cells in display
Figure 2.
Illustration of detected chromosomes in WG-59 immortalized cultured cells in display colors,
colors, showing a pseudotriploid cell line with several aberrations.
showing a pseudotriploid cell line with several aberrations.
The sample WG-59 cell line was derived from WG-59 donor tissue, with full authentication
(Table
2). Furthermore,
using
a detection
limit of donor
1:105, no
mitochondrial
from stated
Thestated
sample
WG-59
cell line was
derived
from WG-59
tissue,
with full sequences
authentication
5
mouse,
rat
or
Chinese
and
Syrian
hamster
cells
could
be
detected
in
WG-59
samples.
The
samples
(Table 2). Furthermore, using a detection limit of 1:10 , no mitochondrial sequences from mouse, rat
were derived from pure human cell cultures. Generated STR profiles of the WG-59 cell line showed
or Chinese
and Syrian hamster cells could be detected in WG-59 samples. The samples were derived
no match with any reference cell line (Figure 3), as indicated by the search of the databases of cell
from pure
human cell cultures. Generated STR profiles of the WG-59 cell line showed no match with
banks ATCC (USA), Japanese Collection of Research Bioresources Cell Bank (Japan), RIKEN (Japan),
any reference
3), as indicated
by the
search
of the databases
of Authenticated
cell banks ATCC
Koreancell
Cellline
Line(Figure
Bank (Korea),
Wistar Institute
(USA),
European
Collection of
Cell (USA),
Japanese
Collection
of Research
Bioresources
Cell Bank (Japan),
RIKEN (Japan),
Cell Line
Cultures
(UK) and
Deutsche Sammlung
von Mikroorganismen
und Zellkulturen
(DSMZ,Korean
Germany).
The
WG-59
cell
line
shows
a
missing
Y-chromosome
in
the
STR
analysis
(Table
2).
Bank (Korea), Wistar Institute (USA), European Collection of Authenticated Cell Cultures (UK) and
Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Germany). The WG-59 cell line
Table 2. Allelic list of the WG-59 cell line and tissue donor STR loci profiling using 8 different
shows a missing
Y-chromosome in the STR analysis (Table 2).
polymorph loci, performed by DSMZ, Germany, showing that these two samples are of the
same origin.
Table 2. Allelic list of the WG-59 cell line and tissue donor STR loci profiling using 8 different
MarkerGermany,
WG-59
Cells that
WG-59
Tissue
polymorph loci, performed by DSMZ,
showing
these
two samples are of the same origin.
D5
D5’
Marker
D13
D5D13’
D5’
D13D7
D13’D7’
D7D16
D7’D16’
D16
vWA
D16’
vWA’
vWA
TH01
vWA’
TH01
TH01’
TH01’
TPOX
TPOX’
CFS1
CSF1’
Amel
Amel’
10
12
WG-59
Cells
11
11 10
12
9 11
10 11
12 9
13 10
12
14
13
15 14
9 15
9.3 9
9.3
8
11
11
11
X
X
10
12
WG-59
Tissue
11
11 10
12
9
11
10 11
9
12
13 10
12
14
13
15 14
9
15
9
9.3
9.3
8
11
11
11
X
Y
Cells 2016, 5, 19
TPOX
TPOX’
CFS1
CSF1’
Amel
Amel’
8
11
11
11
X
X
8
11
11
11
X
Y
7 of 14
(A)
(B)
Figure 3. Electropherogram of the short tandem repeat (STR) profiling of (A) the WG-59 cell line
Figure 3. Electropherogram of the short tandem repeat (STR) profiling of (A) the WG-59 cell line and
and
(B)WG-59
the WG-59
donor
tissue.
(B) the
donor
tissue.
3.3. Flow Cytometry Analysis of the WG-59 Cell Line
3.3. Flow Cytometry Analysis of the WG-59 Cell Line
The purity of isolated cells was analyzed by flow cytometry. The cells expressed the typical
The purity of isolated cells was analyzed by flow cytometry. The cells expressed the typical VSMC
VSMC proteins, e.g., α smooth muscle actin and myosin heavy chain. As shown in Figure 4A–C,
proteins, e.g., α smooth muscle actin and myosin heavy chain. As shown in Figure 4A–C, 84.4% of
84.4% of SMCs were double positive for α smooth muscle actin and myosin heavy chain; 86.5%
SMCs were double positive for α smooth muscle actin and myosin heavy chain; 86.5% were positive
were positive for α smooth muscle actin and calponin; and 83.9% for myosin heavy chain (SM22)
for α smooth muscle actin and calponin; and 83.9% for myosin heavy chain (SM22) and calponin;
and calponin; whereas negative cells accounted for 2.98%, 2.07% and 5.66%, respectively. WG-59
whereas negative cells accounted for 2.98%, 2.07% and 5.66%, respectively. WG-59 cells showed an
cells showed an almost lack of smoothelin expression, as already observed by Grewal et al. [7] and
almost lack of smoothelin expression, as already observed by Grewal et al. [7] and declared typical for
declared typical for VSMC isolated from BAV patients (Figure 4G,H).
VSMC isolated from BAV patients (Figure 4G,H).
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14
88of
Figure 4. Purity analysis of isolated VSMCs with flow cytometry showing α-actin and myosin heavy
Figure
4. Puritycells
analysis
isolated
VSMCs
with flowconfluency.
cytometry showing
α-actin
and(B)
myosin
heavy
chain-positive
at theof20th
passage
and 80%–90%
(A) Isotype
control;
WG-59
cells;
chain-positive
cells
at
the
20th
passage
and
80%–90%
confluency.
(A)
Isotype
control;
(B)
WG-59
(C–D) purity analysis of isolated VSMCs with flow cytometry showing α-actin- and calponin-positive
cells;
(C–D)
purity
analysis
of isolated
VSMCs(E–F)
withpurity
flowanalysis
cytometry
showing
α-actinand
cells at
the 20th
passage
and 80%–90%
confluency;
of isolated
VSMCs
with flow
calponin-positive
cells
at
the
20th
passage
and
80%–90%
confluency;
(E–F)
purity
analysis
of
isolated
cytometry showing myosin heavy chain- and calponin-positive cells at the 20th passage and 80%–90%
VSMCs
with (G–H)
flow cytometry
showing
myosinVSMCs
heavy chainandcytometry
calponin-positive
at the 20th
confluency;
purity analysis
of isolated
with flow
showingcells
smoothelin
and
passage
and
80%–90%
confluency;
(G–H)
purity
analysis
of
isolated
VSMCs
with
flow
cytometry
the isotype control of WG-59 cells at the 20th passage and 80%–90% confluency. Histogram
plot of
showing
smoothelin
and
isotype
control
of WG-59
at the 20th passage and 80%–90%
Smoothelin
expression
(G).the
Isotype
control
in pseudo
colorcells
plot (H).
confluency. Histogram plot of Smoothelin expression (G). Isotype control in pseudo color plot (H).
3.4. Cell Contraction Assay
3.4. Cell Contraction Assay
Using the cell contraction assay, we aimed to visualize the contraction ability of WG-59 cells.
Using the cell contraction assay, we aimed to visualize the contraction ability of WG-59 cells.
The size of the collagen gel matrix was measured at the beginning and end of the assay. However,
The size of the collagen gel matrix was measured at the beginning and end of the assay. However, no
size change could be detected at any time during the assay incubation period of seven days after
stress release.
Cells 2016, 5, 19
9 of 14
Cells
2016,change
5, 19
no size
of 14
could be detected at any time during the assay incubation period of seven days9after
stress release.
3.5. Gene Expression Analysis of the TGFβ Pathway
3.5. Gene Expression Analysis of the TGFβ Pathway
Various gene expressions of the TGFβ pathway were significantly different in losartan-treated
Various
gene expressions
the TGFβ
pathway
significantly
different
losartan-treated
VSMCs
compared
to untreatedofcells.
However,
the were
expression
differences
wereinlow;
none of the
VSMCs
compared
to
untreated
cells.
However,
the
expression
differences
were
low;
of the
significant changes were over two-fold (Figure 5). The scatter plot showed a positive none
and strong
significant changes
over two-fold
5). The scatter
plotAmong
showedthe
a positive
and strong
correlation
between were
losartan-treated
cells(Figure
and non-treated
culture.
genes showing
the
correlation
between
losartan-treated
cells
and
non-treated
culture.
Among
the
genes
showing
the
most significant expression changes were endoglin (1.14-fold downregulation after losartan
most significant
expression
changes were
endoglin
(1.14-fold downregulation
losartanptreatment;
treatment;
p = 0.0052)
and CDC25A
(1.24-fold
downregulation
after losartanafter
treatment;
= 0.0008,
p = 0.0052)
Table
3). and CDC25A (1.24-fold downregulation after losartan treatment; p = 0.0008, Table 3).
Figure
the signal
signal intensities
intensities of
of TGFβ-pathway
TGFβ-pathway genes.
Figure 5.
5. Correlation
Correlation between
between the
genes. Expression
Expression analysis
analysis in
in
losartan-treated
and
untreated
cells.
Cells
treated
with
losartan
are
plotted
against
untreated
losartan-treated and untreated cells. Cells treated with losartan are plotted against untreated cells.cells.
The
The
scatter
shows
a positive
tendency
a strong
correlation
between
the samples.
two samples.
scatter
plot plot
shows
a positive
tendency
withwith
a strong
correlation
between
the two
Table
Significantlyregulated
regulatedgenes
genes
WG-59
after
losartan-treatment
compared
to untreated
in in
WG-59
after
losartan-treatment
compared
to untreated
cells.
Table 3.3. Significantly
cells.
−,
downregulation;
+,
upregulation.
´, downregulation; +, upregulation.
Fold Change
t-Test
t-Test
1 µM Losartan
Description
1 µM Losartan
p-Value
Gene
Gene Bank
vs. Control p-Value
vs. Control
ACVR2
Activin
typeIIA
IIA
NM_001616.3
0.0274
ACVR2
ActivinAAreceptor,
receptor, type
NM_001616.3
´1.16−1.16
0.0274
BMP and activin
membrane-bound
BMP and
activin
BAMBI
NM_012342.2
0.0273
BAMBI
NM_012342.2
´1.10−1.10
0.0273
inhibitor
membrane-bound inhibitor
BMP4
Bone morphogenetic
protein 4
NM_001202.3
−1.12
0.0325
Bone morphogenetic
BMP4
NM_001202.3
´1.12
0.0325
4
BMPER
BMP binding protein
endothelial
regulator
NM_133468
−1.16
0.0121
BMP binding
CDC25A
Cell division
cycle 25A
NM_001789.2
0.0008
BMPER
NM_133468
´1.16−1.24
0.0121
endothelial
regulator
CST3
Cystatin
C
NC_000020.10
−1.11
0.0305
CDC25A
NM_001789.2
´1.24
0.0008
Cell
division
cycle
25A
ENG
Endoglin
NM_000118
−1.14
0.0052
Cystatin C
NC_000020.10
´1.11−1.13
0.0305
FST CST3
Follistatin
NM_013409
0.0099
ENG
NM_000118
´1.14
0.0052
Endoglin
GDF5
Growth Differentiation Factor 5
NM_000557
−1.26
0.0091
NM_013409
´1.13−1.06
0.0099
Follistatin
JUN FST
Jun Proto-Oncogene
NM_002228
0.0479
GrowthActivator,
Differentiation
PLAUGDF5 Plasminogen
urokinase NM_000557
NM_002658
0.0052
´1.26−1.07
0.0091
Factor 5
SMAD3
SMAD family member 3
NM_005902
1.01
0.0335
JUN
Jun Proto-Oncogene
NM_002228
´1.06
0.0479
SOX4
SRY (sex determining region Y)-box 4
NM_003107
−1.25
0.0061
Signal transducer and activator of
STAT1
NM_007315
−1.08
0.0015
transcription, 1.91 kD
TGFB1
Transforming growth factor beta 1
NM_000660
1.02
0.0460
TGFBI
Transforming growth factor,
NM_000358
−1.12
0.0316
Gene
Description
Gene Bank
Fold Change
Cells 2016, 5, 19
10 of 14
Table 3. Cont.
Fold Change
t-Test
Gene
Description
Gene Bank
1 µM Losartan
vs. Control
p-Value
PLAU
Plasminogen Activator,
urokinase
NM_002658
´1.07
0.0052
SMAD3
SMAD family member 3
NM_005902
1.01
0.0335
SOX4
SRY (sex determining
region Y)-box 4
NM_003107
´1.25
0.0061
STAT1
Signal transducer and
activator of
transcription, 1.91 kD
NM_007315
´1.08
0.0015
Transforming growth
factor beta 1
NM_000660
1.02
0.0460
´1.12
0.0316
Cells 2016, 5, 19
TGFB1
TGFBI
TGFBR1
TGFBR1
HPRT1
HPRT1
Transforming
growth
factor,
beta-induced,
68 kDa
NM_000358
beta-induced,
68
kDa
Transforming growth factor beta,
Transforming
receptorgrowth
1
factor beta, receptor 1
Hypoxanthine
Hypoxanthine
phosphoribosyltransferase1
phosphoribosyltransferase1
NM_004612
NM_004612
NM_000194
NM_000194
−1.20
´1.20
−1.04
´1.04
10 of 14
0.0028
0.0028
0.0396
0.0396
Endoglin and CDC25A expressions were measured after treating cells with three different
Endoglin and
CDC25A
expressions
were
measured
afteranalysis
treatingconfirmed
cells withgene
threeexpression
different
concentrations
of losartan
(Figure
6). Single
real-time
RT-PCR
concentrations
of
losartan
(Figure
6).
Single
real-time
RT-PCR
analysis
confirmed
gene
expression
array
array results. Endoglin was downregulated in both measurements. Increasing the losartan
results.
Endoglin
was
downregulated
in
both
measurements.
Increasing
the
losartan
concentration
concentration caused downregulation of endoglin expression, but not significantly.
caused downregulation of endoglin expression, but not significantly.
Figure 6.6.Single
RT-PCR
of endoglin
after WG-59
increasing
concentrations.
Figure
Singlereal-time
real-time
RT-PCR
of endoglin
aftertreatment
WG-59 with
treatment
withlosartan
increasing
losartan
Increasing the losartan
concentration
not downregulate
significantly. endoglin
concentrations.
Increasing
the could
losartan
concentrationendoglin
could expression
not downregulate
expression significantly.
4. Discussion
4. Discussion
TAAD is estimated to occur in three cases per 100,000 individuals per year and is a major cause
of death [14]. Some publications define TAAD by medial degeneration characterized by VSMC
TAAD is estimated to occur in three cases per 100,000 individuals per year and is a major cause
loss, fragmentation and depletion of elastic fibers, as well as accumulation of proteoglycans and
of death [14]. Some publications define TAAD by medial degeneration characterized by VSMC loss,
glycosaminoglycans within cell-depleted aorta tunica media areas [15]. Due to the role of VSMCs
fragmentation and depletion of elastic fibers, as well as accumulation of proteoglycans and
in TAAD, it is crucial to investigate the potential contribution of VSMC to TAAD development. An
glycosaminoglycans within cell-depleted aorta tunica media areas [15]. Due to the role of VSMCs in
immortalized VSMC cell line could be an essential and powerful tool for this investigation.
TAAD, it is crucial to investigate the potential contribution of VSMC to TAAD development. An
In this study, we generated an immortalized VSMC cell line, WG-59, isolated from the aorta of a
immortalized VSMC cell line could be an essential and powerful tool for this investigation.
BAV patient. The WG-59 cell line showed a missing Y-chromosome in STR analysis. The phenomenon
In this study, we generated an immortalized VSMC cell line, WG-59, isolated from the aorta of a
BAV patient. The WG-59 cell line showed a missing Y-chromosome in STR analysis. The
phenomenon of uniparental disomy is sometimes observed during cell line establishments and is
most probably due to loss of function mutations within DNA repair genes. Connected to
bottlenecking outgrowths of sub-lines, these modifications can become visible.
Cells 2016, 5, 19
11 of 14
of uniparental disomy is sometimes observed during cell line establishments and is most probably due
to loss of function mutations within DNA repair genes. Connected to bottlenecking outgrowths of
sub-lines, these modifications can become visible.
Infection of human cells by SV-40 alters the cells and leads to characteristic changes.
Transformation can be defined by expression of SV-40 large T-antigen, loss of contact inhibition,
enhanced growth potential and growth in low-serum media [16]. Immortalized cell cultures usually
enter a “crisis” characterized by reduced proliferation, loss of attachment to the flask and the
development of multinucleated or giant cells [17]. Cultures surviving this crisis show very high
growth capacity and provide an excellent working base for long-term experiments. To confirm our
immortalized WG-59 cell culture containing VSMC, cell shape was observed daily by microscopy, and
flow cytometric measurements were performed.
Cytogenetic studies of WG-59 cells showed polyploid karyotypes with complex rearrangements,
loss of different autosomal chromosomes and both Y-chromosomes. The karyotype instability of the
Y-chromosomes in cells is typical for the loss of the small chromosomes in rapidly-proliferating cells
similar to immortalized SV-40 cell lines [13]. This might be the reason for the alteration in the cytogenic
profile of the cell culture, e.g., loss of the amelogenin marker in STR analysis. The telomeric associations
of the acrocentric chromosomes characteristic for SV-40 transformed cells were also found [18].
The expression of typical VSMC α smooth muscle cell actin, myosin heavy chain and calponin was
detected in more than 95% of the cells (single- and double-positive cells), indicating that the culture
consisted mainly of VSMCs. Other than VSMCs of TAV patients, VSMCs isolated from BAV patient
tissue lack smoothelin expression, as shown by Grewal et al. [7] and confirmed in the present study.
The VSMCs phenotype varies from quiescent and contractile to proliferative and synthetic to
execute a diversity of functions. Unlike the contractile phenotype, the synthetic phenotype possesses
a high proliferative index and a low expression of contractile apparatus proteins [19]. In vitro, the
synthetic VSMC phenotype tends to grow in a hill and valley morphology, which has been observed
in previous studies of WG-59 VSMC cultures conducted by this group [20]. Thus, the hill and valley
morphology observed in these cultured cells may be due to the fact that WG-59 cells are of a synthetic
and proliferative phenotype and therefore express only a low amount of contractile proteins. This
would explain the fact that no observable change in the collagen matrix size or in the contractility of
the WG-59 cell culture in the cell contraction assay was detected. This would explain the fact that
we were not able to observe any collagen size change and therefore contractility of WG-59 in the cell
contraction assay.
The exact mechanisms leading to aortic aneurysms in the ascending aorta of bicuspid aortic valve
(BAV) are poorly understood. It is unclear whether aortic changes in patients with BAV (large aortic
diameter, reduced arterial elasticity and increased degeneration of the aortic media layer) relate to
hemodynamic forces due to aortic valve dysfunction or whether these abnormalities result from an
aortic wall weakness caused by genetic defects [21]. Several BAV studies have revealed high rates of
VSMC apoptosis [22,23], similar to those observed in MFS, even in the non-dilated aorta [24]. MFS
showed overexpression of TGFβresulting from a mutation of the FIBRILLIN-1 gene [25]. On the
other hand, in tissues obtained from patients with LDS, there is an apparent paradoxical increase in
canonical TGFβ signaling with an increased expression of SMAD2, CTGF and collagen. The opposing
mechanisms underlying this unexpected and paradoxical effect are unclear. However, the situation is
the same in LDS as that in BAV-associated aortopathy. Losartan, an AT1 R antagonist, was suggested
to reduce aortic dilation rates at least in a small pediatric cohort of MFS patients [26]. In the present
study, the effect of losartan on the TGFβ signaling pathway was investigated due to the similarity of
aortic dilation to those in MFS and LDS.
In addition to the genes TGFβ1, TGFβR1 and SMAD3, the gene encoding auxiliary receptor for
TGFβ endoglin was significantly downregulated after losartan treatment. Endoglin is involved in
angiogenesis and vessel homoeostasis; an endoglin gene mutation leads to hereditary hemorrhagic
telangiectasia [27]. Endoglin regulates the expression and activation of endothelial nitric oxide synthase,
Cells 2016, 5, 19
12 of 14
which is believed to play a role in angiogenesis and in the composition of vascular resistance in
aneurysm formation [28]. Endoglin plays a pivotal role in the differentiation of neural crest cells into
the VSMC that populate the aorta. Mancini et al. demonstrated that in endoglin ´ /´ mice, defective
VSMC development occurs, which leads to the assumption that endoglin plays an important role in
vessel wall composition on aneurysm formation [29]. On the other hand, it has been shown that TGFβ
and endoglin expressions are both elevated in cultured VSMCs derived from MFS patients. Losartan,
an AT1 R blocker, prevents aneurysm progression in MFS by decreasing TGFβ activity [30,31]. In our
study, the expression of TGFβ and endoglin was decreased after treating a VSMC culture with losartan.
This indicates the beneficial effects of losartan on the overexpression of TGFβ signaling pathway
proteins. Whether the differences in TGFβ and endoglin levels in BAV patients are some of the causes
of aneurysm development in BAV is unclear. Further investigations are warranted to clarify the effect
of these proteins. A previous study showed direct dysregulation caspase-3 signaling in VSMCs isolated
from BAV patients [20]. We are planning to induce apoptosis in primary VSMCs and to investigate the
caspase-3 activity and signaling pathway in those cells in comparison with immortalized WG-59 cells.
As the next step, we are planning on silencing important pathways (fibroblast growth factor, caspase-3
and endoglin [32]) for specific studies to improve our understanding of the mechanisms associated
with apoptosis signaling in BAV.
5. Study Limitation
We are aware of the fact that we only present the generation of one cell line in this study and that
multiple chromosomal abnormalities in our cell line may affect the results and limit the comparability
of these cells with other primary cell culture. An update of the literature, however, scored no results
regarding the generation of human VSMC culture isolated from BAV patients, enabling investigation
of BAV ascending aorta.
Losartan should be metabolized to gain an activated form available for the organism. Telmisartan
would be more suitable for in vitro usage. The application of telmisartan as an activated form of
angiotensin II blocker, which on its part activates eNOS, will be one of our future projects. Telmisartan
is known to decrease inflammatory protein expression, such as C- reactive protein in coronary plaque
components [33]. However, according to our experience and previous results, only low inflammatory
processes are detected in ascending aorta of BAV [34,35].
Acknowledgments: The authors would like to acknowledge and thank the Leducq Fondation for supporting our
work and giving us the opportunity to study BAV aortopathy over the coming years. Furthermore, we would like
to thank the team of Department of Cardiac and Thoracic Vascular Surgery, Luebeck, Germany, as well as Merck
Research Laboratories (New Jersey, USA) for supplying us with losartan.
Author Contributions: S.A.M conceived and designed the experiments; S.A.M., G.B., T.F., A.R., P.L-K. and D.S-B.
performed the experiments; S.A.M. and J.Y. analyzed the data; P.R. contributed with software tools; S.A.M. P.L-K.
and D.S-B wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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